Assays and, in particular, immunoassays are widely used as diagnostic tools in bacterial, viral and parasitic diseases as well as infectious diseases such as AIDS which constitute major health problems around the world.
Immunoassays are generally utilized for detecting and/or quantifying the amount of analyte in serum or other biological fluids and are based principally on the binding of specific binding substance, such as an antibody, to a particular analyte which might be present in a specimen. Unfortunately, detection of analyte is often hindered by the presence of endogenous binding substances because analyte is masked by the formation of endogenous binding substance-analyte complexes. For example, analytes such as Human Immunodeficiency Virus (HIV)-1 p24 core protein are present in serum, plasma, etc. bound in equilibrium to human antibody that has been produced as a consequence of viral infection. Masking by antibody (due to formation of antigen-antibody complexes) interferes with detection of analyte in a bound state by conventional methods such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Consequently, only free analyte can be detected. However, determination of the amount of free analyte will be affected by a variety of factors such as the concentration of analyte, and the concentration and affinity of the antibody.
The inability of current methods to detect total concentration of analyte creates a serious problem where measurement of total analyte is important. For example, measurement of HIV antigen levels in sera or plasma of HIV-infected individuals is important in determining the existence of antigen or infectious virus before seroconversion and for prognosis and is a useful tool for monitoring antiviral drug therapy. Diagnosis of HIV infection in children during the first year of life is hampered by the inability of standard serologic assays to diagnose HIV due to persisting maternally derived antibody. Thus, to understand the role of HIV in the process of disease development, control and prognosis, reliable measurement of serum antigen and viral antigen production in HIV-infected individuals is very important.
Efforts to increase sensitivity and specificity of assay systems to improve detection and/or quantification of analyte have been undertaken as illustrated by the following:
U.S. Pat. No. 4,604,348, issued to Neurath on Aug. 5, 1986, describes a process for detecting antibodies or antigens in a sample where the antigens or antibodies are present in the form of an immune complex. This process involves irreversibly attaching the antigen or antibody to a protein sorbing solid support in the presence of a dissociating buffer. Once antigen or antibody derived from immune complex has been adsorbed to the solid support, the existence thereof can be determined by RIA or ELISA.
Similarly, Neurath et al., Proc. Natl. Acad. Sci. USA, 79: 4415-4419 (July 1982), describe a solid-phase method for separating antigens from antibodies. Specifically, immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Detection is then effected using RIA.
European Patent Application Publication No. 0 155 104 published Sep. 18, 1985 describes a free analyte assay in which analyte is present partly bound to natural protein binders and in which free analyte and an analyte derivative compete for reaction with a specific binder. A differential blocking agent is used to reduce binding of the analyte derivative but without reducing binding to analyte to natural protein binders.
U.S. Pat. No. 4,703,001, issued to Vodian et al. on Oct. 27, 1987, describes an immunoassay for detecting serum analytes using pH dependent chaotropic acids so that the analyte is substantially free from its antibody and/or other serum proteins are largely denatured.
Nishanian et al., J. Infect. Dis., 162: 21-28 (July 1990), von Sydow et al., Br. Med. J., 296: 238-240 (January 1988), and Kageyama et al., J. Virol. Meth., 22: 125-131 (1988), describe methods to enhance detection of HIV-1 antigen (present in immune complexes) in serum or plasma samples by pretreating samples at an acidic pH to dissociate immune complexes and denatured antibodies with little or no compromise of antigen immunoreactivity.
While these methods facilitate detection of undetectable or poorly detectable analytes, they do not provide for determination of total analyte concentration.